Single Cell Detection of the p53 Protein by Mass Cytometry

Abstract

Purpose: The p53 protein and its post-translational modifications are distinctly expressed in various normal cell types and malignant cells and are usually detected by immunohistochemistry and flow cytometry in contemporary diagnostics. Here, we describe an approach for simultaneous multiparameter detection of p53, its post-translational modifications and p53 pathway-related signaling proteins in single cells using mass cytometry. Method: We conjugated p53-specific antibodies to metal tags for detection by mass cytometry, allowing the detection of proteins and their post-translational modifications in single cells. We provide an overview of the antibody validation process using relevant biological controls, including cell lines treated in vitro with a stimulus (irradiation) known to induce changes in the expression level of p53. Finally, we present the potential of the method through investigation of primary samples from leukemia patients with distinct TP53 mutational status. Results: The p53 protein can be detected in cell lines and in primary samples by mass cytometry. By combining antibodies for p53-related signaling proteins with a surface marker panel, we show that mass cytometry can be used to decipher the single cell p53 signaling pathway in heterogeneous patient samples. Conclusion: Single cell profiling by mass cytometry allows the investigation of the p53 functionality through examination of relevant downstream signaling proteins in normal and malignant cells. Our work illustrates a novel approach for single cell profiling of p53.

Probe‐based mass spectrometry approaches for single‐cell and single‐organelle measurements

“…Although, MALDI MS does generate intact molecular proteins allowing putative protein identification based on accurate mass, it may be limited by the low cellular abundance of proteins and would benefit from the development of protein specific tags or chemical derivation agents that would improve the ionization efficiency of the proteins of interest (Fülöp et al, 2020). This approach was used for determining the protein expression of the p53 protein in cancer cells by staining the cells with heavy metal isotope labeled antibodies for subsequent detection using mass cytometry (Fagerholt et al, 2020).…”

“…Although, MALDI MS does generate intact molecular proteins allowing putative protein identification based on accurate mass, it may be limited by the low cellular abundance of proteins and would benefit from the development of protein specific tags or chemical derivation agents that would improve the ionization efficiency of the proteins of interest (Fülöp et al, 2020). This approach was used for determining the protein expression of the p53 protein in cancer cells by staining the cells with heavy metal isotope labeled antibodies for subsequent detection using mass cytometry (Fagerholt et al, 2020). Combining liquid microjunction extraction techniques to desorption electrospray ionization mass spectrometry (DESI) has led the development of nanospray DESI (nanoDESI) which has demonstrated the label‐free detection of proteins in tissue sections (Feider et al, 2016; Griffiths et al, 2017; Hsu et al, 2015).…”

A comprehensive study of p53 protein

“…Mass cytometry is a relatively new technique that uses mass spectrometry detection along with metal isotope‐bound antibodies to quantify up to 50 properties of each cell. When analyzing cancer patient samples, it is crucial to consider the expression of the p53 protein and its signaling pathway, according to Fagerholt et al 262 who focused on the use of mass cytometry in the evaluation of hematological malignancies. The investigation of primary patient sample p53 using mass cytometry has enormous potential.…”

Antifouling Electrochemiluminescence Biosensor Based on Bovine Serum Albumin Hydrogel for the Accurate Detection of p53 Gene in Human Serum

“…p53 protein is widely recognized as a key tumor suppressor with a critical function in the protection of cellular integrity from various forms of genotoxic stress, including DNA damage, oncogenic activation, and telomere erosion. − However, mutations in p53 can result in losing of its tumor suppressor functions and the manifestation of oncogenic activities, characterized by the disturbance of cellular transcriptome and proteome, the promotion of tumorigenesis, the induction of chemoresistance, and the enhancement of metastatic potential. As such, the mutant p53 protein has emerged as a potent oncogenic driver of cancer cell proliferation, invasion, and dissemination, attracting significant attention in many studies. − …”